1. Volume 27, Issue 11, Pages 3107-3116.e3 (June 2019)
Presynaptic Expression of LRIT3 Transsynaptically Organizes the Postsynaptic Glutamate Signaling Complex Containing TRPM1 
Nazarul Hasan, Gobinda Pangeni, Catherine A. Cobb, Thomas A. Ray, Emily R. Nettesheim, Kristina J. Ertel, Daniel M. Lipinski, Maureen A. McCall, Ronald G. Gregg 
Cell Reports 
Volume 27, Issue 11, Pages e3 (June 2019)
DOI: /j.celrep
Copyright © 2019 The Author(s) Terms and Conditions

2. Cell Reports 2019 27, 3107-3116.e3DOI: (10.1016/j.celrep.2019.05.056)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1 LRIT3 Is Expressed in Rods
(A) In situ hybridization for mRNA probes in transverse retinal sections shows that the positive control, Pol2r, is ubiquitously expressed throughout all retinal layers. Grm6 expression is found in the outer part of the inner nuclear layer (INL), where the cell bodies of the ON BCs are located. Dab, the negative control shows no expression; Lrit3 expression is found almost exclusively in the outer nuclear layer (ONL), where the photoreceptor cell bodies are located. GCL, ganglion cell layer. DAPI stain nuclei (blue) and mRNA probes (red). Scale bar, 20 μm.
(B) Schematic diagram of the rAAV RHO::Lrit3 vector (top) and the time course of treatment and analyses (bottom).
(C) Transverse retinal sections of control (i), Lrit3−/− (ii), and rAAV RHO::Lrit3-treated Lrit3−/− (iii) retinas stained for mGluR6 (green) and LRIT3 (magenta). Arrowheads in (i) indicate cone terminal staining. OPL, outer plexiform layer. Scale bar, 10 μm.
(D) Whole mounts imaged at the level of the OPL from control (i) and rAAV RHO::Lrit3-treated Lrit3−/− (ii) retinas stained for mGluR6 (green) and LRIT3 (magenta). Dotted circles in (i) indicate cone terminals. Boxed region is enlarged below. Note the absence of cone terminal staining in the rAAV RHO::Lrit3-treated Lrit3−/− retina (ii).
(E) Quantification of the percentage of rod synapses expressing LRIT3 in control, Lrit3−/−, and rAAV RHO::Lrit3-treated Lrit3−/− (n = 5 eyes, 5 mice).
Cell Reports  , e3DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

4. Figure 2
LRIT3 Expression in Lrit3−/− Rods Restores Expression of TRPM1 to the Dendrites of RBCs in the Outer Plexiform Layer
(A) Transverse retinal sections of control (i), Lrit3−/− (ii), and rAAV RHO::Lrit3-treated Lrit3−/− (iii) retinas stained for TRPM1 (green) and LRIT3 (magenta). Scale bar, 10 μm.
(B) Whole mount imaged at the level of the OPL of a retina from a rAAV RHO::Lrit3-treated Lrit3−/− mouse stained for mGluR6 (green), LRIT3 (red), and TRPM1 (magenta). Note that TRPM1 expression co-localizes with both LRIT3 and mGluR6 but is absent when LRIT3 is absent. Scale bar, 20 μm.
(C) Schematic of the normal postsynaptic elements in RBPs, indicating an unknown location for LRIT3, the impact on this complex of the loss of LRIT3, and the consequence of restoring LRIT3 expression to rod photoreceptors.
Cell Reports  , e3DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

5. Figure 3 LRIT3 Expression in Lrit3−/− Rods Restores RBC Function
(A and B) Scotopic (A) and photopic (B) electroretinograms at different stimulus intensities for one control eye (black), an age-matched Lrit3−/− eye (red), and an rAAV RHO::Lrit3-treated Lrit3−/− eye (blue).
(C and D) Average stimulus-response plots for the ERG b-wave amplitude under scotopic (C) and photopic (D) conditions for control rAAV RHO::GFP (n = 3, black), Lrit3−/− (n = 3, red), and rAAV RHO::Lrit3-injected Lrit3−/− (n = 8, blue). Pale lines are the responses for individual eyes, and dark lines are the mean ± SD for each group. The dashed red and blue lines indicate no b-wave (zero amplitude) in these animals.
Statistics: (C) comparison of control versus rAAV RHO::LRIT3 groups, ∗∗padj < 0.01 for stimuli, two-way repeated-measures ANOVA; comparison of rAAV RHO::LRIT3-injected Lrit3−/− versus Lrit3−/− groups (b-wave amplitude = 0), ∗padj < 0.03 for all stimuli compared using single-sample t test adjusted for multiple comparisons by Bonferroni correction. There are no differences in a-wave amplitude between groups under either scotopic or photopic conditions (two-way repeated-measures ANOVA, padj > 0.05). padj, adjusted p value.
Cell Reports  , e3DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

6. Figure 4
LRIT3 Expression in Rods Restores Normal ON RGC Function in Lrit3−/− Retinas
(A) Example of raster plots and peri-stimulus time histograms (PSTH) of each cell type as indicated, recorded on the MEA (multi-electrode array).
(B) Frequency distribution of time to peak (TTP) for the ON responses in control (i), Lrit3−/− (ii), and rAAV RHO::Lrit3-treated Lrit3−/− (iii) retinas.
(C) Percentage of each cell type recorded under scotopic (i) and photopic (ii) conditions. dON and dON/OFF cells are omitted because of their small numbers, but see Table S1 for numbers. All groups were analyzed using chi-square tests, and when significant (p < 0.05) two-way post hoc tests were done (chi-square) with Bonferroni adjustment of the p values for multiple testing (∗padj < 0.025).
(D and E) Example PSTH’s for a cell from control (Di) and rAAV RHO::Lrit3 retinas (Ei) in Ringer’s solution, in Ringer’s plus 5 μM L-AP4, and after L-AP4 washout. (Dii) The peak responses for individual cells (gray lines) and mean ± SEM (red lines) of control (Dii) and rAAV RHO::Lrit3-treated Lrit3−/− (Eii) retinas are shown. The response amplitudes in (D) and (E) in L-AP4 was significantly decreased (∗padj < , repeated-measures ANOVA, Bonferroni corrected for both datasets) and after washout was not significant (ns; padj > 0.4). padj, adjusted p value.
Cell Reports  , e3DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions

7. Figure 5
LRIT3 Expression in Rods of Adult Lrit3−/− Mice Restores RBC Function
(A) Whole mount of retinas from P35 rAAV RHO::Lrit3-treated Lrit3−/− mice stained for LRIT3 (magenta) and TRPM1 (green) analyzed at P70. A sample of the LRIT3 puncta is highlighted (dashed circles), showing that they co-localize with TRPM1. Every LRIT3 puncta co-localizes with TRPM1 puncta. Scale bar, 2 μm.
(B) Quantification of the percentage of rod synapses expressing LRIT3 and TRPM1 in WT (black symbol), rAAV RHO::Lrit3-injected animals at P5 (green symbol) or P35 (blue symbol), and Lrit3−/− retinas (red symbol). Note that the P5 data are replicated from Figure 1.
(C and D) Scotopic (C) and photopic (D) electroretinograms at different stimulus intensities for one control eye (black), an age-matched Lrit3−/− eye (red), and an rAAV RHO::Lrit3-treated Lrit3−/− P35 mouse eye (blue).
(E) Scotopic responses to a −3.6 log cd s/m2 stimulus for a control rAAV RHO::GFP (black), a Lrit3−/− (red), and five rAAV RHO::Lrit3-injected P35 Lrit3−/− (blue) mouse eyes.
(F) Response amplitudes from the five animals in (E) (blue triangles) and WT (black circles) to the −3.6 log cd s/m2 stimuli. The red line indicates no b-wave (zero amplitude) in the Lrit3−/− animals (mean ± SD are indicated. The WT data are from Figure 3C.
Statistics: (F) rAAV RHO::Lrit3-injected Lrit3−/− eyes show significantly greater b-waves than Lrit3−/− groups (b-wave amplitude = 0), ∗p < 0.01 (single-sample t test). rAAV RHO::Lrit3-injected Lrit3−/− eyes show significantly smaller b-waves than WT eyes, ∗∗p < (two-sided t test).
Cell Reports  , e3DOI: ( /j.celrep )
Copyright © 2019 The Author(s) Terms and Conditions